Peg3/Pw1 is involved in p53-mediated cell death pathway in brain ischemia/hypoxia.

Emerging evidence has shown that tumor suppressor p53 expression is enhanced in response to brain ischemia/hypoxia and that p53 plays a critical role in the cell death pathway in such an acute neurological insult. However the mechanism remains unclear. Recently it was reported that Peg3/Pw1, originally identified as a paternally expressed gene, plays a pivotal role in the p53-mediated cell death pathway in mouse fibroblast cell lines. In this study, we found that Peg3/Pw1 expression is enhanced in peri-ischemic neurons in rat stroke model by in situ hybridization analysis, where p53 expression was also induced by immunohistochemical analysis. Moreover, we found that p53 was co-localized with Peg3/Pw1 in brain ischemia/hypoxia by double staining analysis. In human neuroblastoma-derived SK-N-SH cells, Peg3/Pw1 mRNA expression is enhanced remarkably at 24 h post-hypoxia, when p53 protein expression was also enhanced at high levels. Subcellular localization of Peg3/Pw1 was observed in the nucleus. Adenovirus-mediated high dose p53 overexpression induced Peg3/Pw1 mRNA expression. Overexpression of Peg3/Pw1 reduced cell viability under hypoxic conditions, whereas that of the C-terminal-deleted mutant and anti-sense Peg3/Pw1 inhibited hypoxia-induced cell death. These results suggest that Peg3/Pw1 is involved in the p53-mediated cell death pathway as a downstream effector of p53 in brain ischemia/hypoxia.     

Computational investigation of the conformational dynamics in Tom20-mitochondrial presequence tethered complexes

The translocase of the outer membrane (TOM) mediates the membrane permeation of mitochondrial matrix proteins. Tom20 is a subunit of the TOM complex and binds to the N-terminal region (ie, presequence) in mitochondrial matrix precursor proteins. Previous experimental studies indicated that the presequence recognition by Tom20 was achieved in a dynamic-equilibrium among multiple bound states of the α-helical presequence. Accordingly, the co-crystallization of Tom20 and a presequence peptide required a disulfide-bond cross-linking. A 3-residue spacer sequence (XAG) was inserted between the presequence and the anchoring Cys residue at the C-terminus to not disturb the movement of the presequence peptide in the binding site of Tom20. Two crystalline forms were obtained according to Ala or Tyr at the X position of the spacer sequence, which may reflect the dynamic-equilibrium of the presequence. Here, we have performed replica-exchange molecular dynamics (REMD) simulations to study the effect of disulfide-bond linker and single amino acid difference in the spacer region of the linker on the conformational dynamics of Tom20-presequence complex. Free energy and network analyses of the REMD simulations were compared against previous simulations of non-tethered system. We concluded that the disulfide-bond tethering did not strongly affect the conformational ensemble of the presequence peptide in the complex. Further investigation showed that the choice of Ala or Tyr at the X position did not affect the most distributions of the conformational ensemble of the presequence. The present study provides a rational basis for the disulfide-bond tethering to study the dynamics of weakly binding complexes.     

Phosphoinositide 3-kinaseγ controls the intracellular localization of CpG to limit DNA-PKcs-dependent IL-10 production in macrophages

Synthetic oligodeoxynucleotides containing unmethylated CpG motifs (CpG) stimulate innate immune responses. Phosphoinositide 3-kinase (PI3K) has been implicated in CpG-induced immune activation; however, its precise role has not yet been clarified. CpG-induced production of IL-10 was dramatically increased in macrophages deficient in PI3Kγ (p110γ(-/-)). By contrast, LPS-induced production of IL-10 was unchanged in the cells. CpG-induced, but not LPS-induced, IL-10 production was almost completely abolished in SCID mice having mutations in DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Furthermore, wortmannin, an inhibitor of DNA-PKcs, completely inhibited CpG-induced IL-10 production, both in wild type and p110γ(-/-) cells. Microscopic analyses revealed that CpG preferentially localized with DNA-PKcs in p110γ(-/-) cells than in wild type cells. In addition, CpG was preferentially co-localized with the acidic lysosomal marker, LysoTracker, in p110γ(-/-) cells, and with an early endosome marker, EEA1, in wild type cells. Over-expression of p110γ in Cos7 cells resulted in decreased acidification of CpG containing endosome. A similar effect was reproduced using kinase-dead mutants, but not with a ras-binding site mutant, of p110γ. Thus, it is likely that p110γ, in a manner independent of its kinase activity, inhibits the acidification of CpG-containing endosomes. It is considered that increased acidification of CpG-containing endosomes in p110γ(-/-) cells enforces endosomal escape of CpG, which results in increased association of CpG with DNA-PKcs to up-regulate IL-10 production in macrophages.     

Synthesis and photophysical properties of zinc myoglobin appending an ethidium ion as a DNA intercalator

In order to elucidate the intramolecular photoinduced electron-transfer or energy-transfer mechanisms of the zinc myoglobin (ZnMb) dyad and to construct a photoreaction system within a Mb–DNA complex, we newly prepared ZnMb appending an ethidium ion (Et⁺). The steady-state fluorescence of ZnMb–Et⁺ at 600 nm and its lifetime (2.2 ns) indicate that the excited singlet state of ¹(ZnMb)* is not quenched by the Et⁺ moiety, whereas the lifetime of the excited triplet state of ³(ZnMb)*–Et⁺ was shorter (τ = 4.3 ms) than those of ZnMb and the intermolecular (ZnMb + ethidium) system. Upon photoirradiation of Et⁺, fluorescence studies indicated the intramolecular quenching reactions from the excited singlet state, ¹(Et⁺)*, to ZnMb, the process of which is likely the photoinduced energy-transfer reaction via a through-space mechanism. We also demonstrate the photophysical and spectroscopic properties of ZnMb–Et⁺ in the presence of calf thymus (CT) DNA. The changes in the absorption and fluorescence spectra of ZnMb–Et⁺ on the addition of CT-DNA up to 15 equiv were very small, indicating that there are no major changes in the heme pocket. However, we observed a longer lifetime of ³(ZnMb)*–Et⁺ in the presence of CT-DNA (τ = 5.3 ms) by single flash photolysis. This was induced by noncovalent interactions between Et⁺ and CT-DNA, followed by a conformational change of Et⁺ at the surface of ZnMb, where the donor–acceptor distance was probably elongated by CT-DNA. The synthetic manipulation at the Mb surface, by using a DNA intercalator coupled with photoinduced reaction, may provide a sensitive transient signal for DNA and valuable information to construct new Mb–DNA complex. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Relationship between scapular position and structural strength of rib cage in quadruped animals

Determining scapular position is a major issue in reconstructing the skeletal systems of extinct quadruped archosaurs and mammals, because the proximal portion of the scapulae has no direct skeletal joint with the vertebrae or ribs. When quadrupeds stand or walk, their trunk is suspended between the forelimbs by the serratus muscles, which arises from the lateral sides of the “thoracic” ribs and inserts into the proximal portion of the costal surface of the scapula. Therefore, the “thoracic” ribs are subjected to a static or dynamic vertical compression between the lifting force from the muscle and the gravitational force from the vertebral column. To investigate the body support function of the ribs, we analyzed the mechanical strength of the ribs of extant tetrapods by the two‐dimensional finite element method, and compared the degree of strength through their craniocaudal scapular positions. The result of this simulation showed that the “thoracic” ribs of quadrupeds, to which the serratus muscles attach, have a relatively higher strength against compaction than the other ribs. In bipeds, however, we did not find a similar correlation between the strength of ribs and the serratus muscle. This implies that the location of robust ribs is associated with the arrangement of the serratus muscle, and provides a probable candidate for determination of the scapular position for extinct quadruped archosaurs and mammals. J. Morphol. 2009. © 2009 Wiley‐Liss, Inc.     

Expression and identification of HERV-W family in Japanese monkeys (Macaca fuscata)

We investigated structural genes (gag, pol, env) of HERV-W family in the Macaca fuscata (Japanese monkey). Those genes are expressed in various tissues (testis, prostate, kidney, cerebellum, thymus, pancreas, intestine, stomach, ovary) of the Japanese monkey in RT-PCR and sequencing analyses. Nine clones for gag, thirty-one clones for pol and thirty-four clones for env fragments of the HERV-W family in monkey tissues were identified and analyzed. These clones showed a high degree of sequence similarity, 82.2-84.7% for gag, 88.4-91.7% for pol, and 90.8-95.4% for env, to those of HERV-W family. Translation to amino acids in all clones derived from the monkey indicated that they showed multiple interruptions of frameshifts and termination codons by deletion/insertion or point mutation. Identical sequences from different tissues of the monkey were found in env and pol clones of the HERV-W family.     

Inter-male mating-like behavior in the domesticated house musk shrew, Suncus murinus

In the present study, inter-male interaction of the domesticated house musk shrew was observed in detail under laboratory conditions. In most cases, during inter-male interaction, male house musk shrews exhibited a sequence of behavior items including tail-wagging, following, mounting and thrusting. In the minority of cases, males did not progress beyond following. Offensive behavior was not sufficiently violent to cause injury. It appeared that role assignment was decided by contact manner and vocalization. One of fundamental characters of this animal made a start of following, in which one shrew followed another, who touched and then separated. Role assignment (i.e., which male led and which followed) was decided in status battle. Roles were often reversed during following. Following behavior was maintained by 'polite' mutual contact, and the interaction progressed to thrusting in the majority cases. After role assignment, the variation in contact manner decreased. The ratio of time spent in front-and-behind contact to that spent in multi-lateral contact increased when both males commenced following behavior simultaneously. This ratio was maintained until the following male snapped after he finished thrusting. Even if the following male did not reach thrusting, he mounted the preceding male. The pairs who did not reach thrusting repeated following behavior or mounting. In those cases, while one male concentrated on touching the other to maintain following, the other attempted to divert attention from the following behavior. Male shrews were able to reach thrusting irrespective of sex.     

Dynamic coupling between the SH2 domain and active site of the COOH terminal Src kinase, Csk

The SH2 domain is required for high catalytic activity in the COOH-terminal Src kinase (Csk). Previous solution studies suggest that a short peptide sequence, the SH2-kinase linker, provides a functional connection between the active site and the distal SH2 domain that could underlie this catalytic phenomenon. Substitutions in Phe183 (tyrosine, alanine, and glycine), a critical hydrophobic residue in the linker, result in large decreases in substrate turnover and large increases in the K(m) for ATP. Indeed, F183G possesses kinetic parameters that are similar to that for a truncated form of Csk lacking the SH2 domain, suggesting that a single mutation disrupts communication between this domain and the active site. Based on equilibrium and stopped-flow fluorescence experiments, the elevated K(m) values for the mutants are due to changes in the rates of phosphoryl transfer and not to reduced ATP-binding affinities. Based on hydrogen-deuterium exchange experiments, glycine substitution reduces flexibility in several polypeptide regions in Csk, tyrosine substitution increases flexibility, and alanine substitution leads to mixed effects compared to wild-type. Normal mode analysis indicates that Phe183 and its environment are under strain, a theoretical finding that supports the results of mutations. Overall, the data indicate that domain-domain interactions, controlled through the SH2-kinase linker, provide a dynamic balance within the Csk framework that is ideal for efficient phosphoryl transfer in the active site.     

Biochemical and medical aspects of the indoleamine 2,3-dioxygenase-initiated L-tryptophan metabolism

Indoleamine 2,3-dioxygenase (EC 1.13.11.42) is a heme-containing dioxygenase which catalyzes the first and rate-limiting step in the major pathway of L-tryptophan catabolism in mammals. Much attention has recently been focused on the dioxygenase because this metabolic pathway is involved not only in a variety of physiological functions but also in many diseases. In this review, the discovery and unique catalytic properties of dioxygenase are described first, and then the recent findings regarding the dioxygenase-initiated tryptophan metabolism are summarized, with special emphasis on the detrimental role of dioxygenase in side effects of interferon-gamma and interleukin-12 (by systemic tryptophan depletion), the escape of malignant tumors from immune surveillance (by immunosuppression caused by tryptophan depletion), several neurodegenerative disorders including Alzheimer's disease (by an aberrant production of neurotoxin, quinolinic acid), and age-related cataract (due to "Kynurenilation," a novel post-translational modification of lens proteins with tryptophan-derived UV filters).